Protease for activating clotting factor VII

ABSTRACT

A protease for activating the blood clotting factor VII, which is inhibited by the presence of aprotinin, is increased in its activity by calcium ions and/or heparin or heparin-related substances, and in SDS-PAGE, on subsequent staining in the non-reduced state, comprises one or more bands in the molecular weight range from 50 to 75 kDa; and in SDS-PAGE, on subsequent staining in the reduced state, comprises a band at 40 to 55 kDa, one or more bands in the molecular weight range from 10 to 35 kDa, and a band in the molecular weight range between 60 and 65 kDa, which corresponds to a proenzyme. Pharmaceutical preparations containing the protease or its proenzyme are suitable for the prophylaxis and treatment of bleeding events, e.g. in the presence of FVIII inhibitors, wound healing and for the treatment of disorders which are caused by fibrin-containing thrombin.

The invention relates to a protease for activating the blood clottingfactor VII, to a process for isolating it, detecting it and inactivatingit, and to medicinal preparations which comprise this protease.

BACKGROUND OF THE INVENTION

The blood clotting system comprises two different, cascade-like pathwaysfor activating clotting factors which are present in the plasma. Theintrinsic or the entrinsic pathway is preferentially used for initiatingclotting, depending on the triggering mechanism.

When a tissue is damaged, thromboplastin (tissue factor, TF withphospholipids) is exposed by the affected cells as the starter of theextrinsic clotting pathway. The membrane-located thromboplastin can bindboth clotting factor VII (FVII) and circulating, activated FVII (FVIIa).In the presence of calcium ions and lipids, this TF-FVIIa complex leadsto the binding of FX, which is converted into its activated form (FXa)by limited proteolysis. FXa in turn leads, by activating prothrombin toform thrombin, to the formation of fibrin and thereby ultimately toclosure of the wound.

While the further activation of the thromboplastin-bound FVII initiallytakes place autocatalytically, in particular, it is supported, after theclotting cascade has been initiated, by FXa and thrombin, in particular,leading to marked reinforcement of the reaction cascade.

The administration of FVIIa or FVIIa-containing concentrates isindicated in certain clinical situations. The so-called FVIII-bypassingactivity (FEIBA) of FVIIa is used in patients who are suffering, forexample, from hemophilia A and have developed antibodies against FVIIIas a consequence of the administration of FVIII. According to presentlyavailable findings, FVIIa is well tolerated in this context and, whileit does not lead to any tendency to thrombosis, it is suitable forensuring that clotting takes place to a limited but adequate extent.Recombinant FVIIa is already being used therapeutically andprophylactically. FVII which has been isolated from blood plasma canalso be activated and then used. Proteases such as thrombin can be usedfor this activation; however, these proteases, as such, can themselvesstrongly activate clotting and lead to the risk of a thrombosis. Forthis reason, subsequent removal or inactivation of thrombin is necessaryand leads to yield losses. As a result of the risk of thrombosis whichis associated with it, the use of FXa or FIIa (thrombin) is frequentlycontraindicated and only indicated in emergencies, e.g. in associationwith extreme loss of blood and unstaunchable hemorrhages.

FVIIa is found in very low concentrations in the plasma of healthysubjects. Only very little is so far known about the formation andorigin of FVIIa which is circulating in the blood. Traces ofthromboplastin which has been expressed or released in association withcell destruction might play a role in this context. Although it is knownthat factor XIIa, for example, can lead to FVII activation under certainconditions, the physiological relevance of this reaction has not yetbeen clarified.

SUMMARY OF THE INVENTION

Surprisingly, a FVII-activating protease, which differs from all thepreviously known proteases, has now been found in connection withfractionating human plasma and certain prothrombin complex concentrates.Investigations into this protease have shown that it exhibits aparticularly high amidolytic activity toward the peptide substrate S2288(HD-isoleucyl-L-prolyl-L-arginine-pNA) from Chromogenix AB, Sweden. Aparticular feature of this protease is that the amidolytic activity isefficiently inhibited by aprotinin. Other inhibitors, such as theantithrombin III/heparin complex, are also suitable for the inhibition.On the other hand, its activity is increased by heparin andheparin-related substances such as heparan sulfate or dextran sulfateand calcium ions. Finally, it has been found that this protease is able,in a manner dependent on time and on its concentration, to convert FVIIinto FVIIa. This reaction, too, is inhibited by aprotinin.

Part of the subject matter of the invention is therefore a protease foractivating the blood clotting factor VII, which

a) is inhibited by the presence of aprotinin,

b) is increased in its activity by calcium ions and/or heparin orheparin-related substances, and

c) in SDS-PAGE, on subsequent staining in the non-reduced state, has oneor more bands in the molecular weight range from 50 to 75 kDa and kDa inthe reduced state has a band at 40 to 55 kDa and one or more bands inthe molecular weight range from 10 to 35 kDa.

In the following text, the activated form of the protease is termed“protease” whereas the non-activated form is termed “proenzyme”.

Further investigations with this protease have shown that, afterenriching or isolation, it suffers from a rapid loss of activity, whichwas observed in a solution containing 20 mM tris, 0.15 M NaCl at a pH of7.5. The addition of albumin at a concentration of 0.1% was not able toprevent the activity of the protease from decreasing by 50% after onehour at room temperature. On the other hand, very good stabilization ofthe protease was observed in a solution which was buffered to a pH of6.5 with 50 mM Na citrate. If no particular stabilizers are added to theprotease solution, no, or only slight, losses in activity are observedif the solution is adjusted to a pH of between 4 and 7.2, preferably toa pH of between 5.0 and 7.0. However, it is expedient to add stabilizersto the solution, with suitable stabilizers, apart from citrate, being,in particular, glutamate, amino acids, such as arginine, glycine orlysine, calcium ions and sugars such as glucose, arabinose or mannose inquantities of 1-200 mmol/l, preferably in quantities of 5-100 mmol/l.Efficient stabilization was also achieved by adding glycols such asethylene glycol or glycerol, with quantities of 5-80% by weight,preferably of 10-60% by weight, being used. The pH of the stabilizedsolution should then be between the pH values 4-9.

While the novel protease, and also the proenzyme, can be obtained byrecombinant DNA methods or by production in e.g. the milk of suitabletransgenic animals, they can in particular be obtained by fractionationof blood plasma or of prothrombin complex (PPSB) concentrates. Thestarting material is then first of all subjected to an anion exchangechromatography, which is followed by an affinity chromatography of theeluate. A heparin which is immobilized on a matrix, or a heparin-relatedsubstance such as heparan sulfate or dextran sulfate, is particularlysuitable for the affinity chromatography. When such a chromatographicmethod is used, the novel protease and /or the proenzyme can beselectively bound and then eluted once again using known methods. Theuse of a spacer is advisable for coupling the ligand to the matrix. Aheparin-lysine matrix has been found to be particularly suitable forisolating the novel protease.

In SDS-PAGE with subsequent staining, the protease which has beenisolated by this method exhibits, in the non-reduced state, one toseveral bands which lie closely together in the molecular weight rangeof 55-75 kDa. Following reduction, one to several bands were observed inthe molecular weight range of 15-35 kDa and one band was observed at40-55 kDa. A further band between 60 and 65 kDa, which after scanningand quantitative evaluation, constituted 5-10% of the total protein,showed that non-activated proenzyme was also present. This result wassupported by appropriate investigations using monoclonal antibodiesagainst this protease. It was therefore concluded that the proenzyme ofthis protease can also be prepared, pasteurized and used by the methodaccording to the invention. Part of the subject matter of the inventionis therefore the proenzyme of the protease for activating blood clottingfactor VII. The proportion of the proenzyme is indicated by the bandbetween 60 and 65 kDa. Corresponding to the amino acid sequence whichconstitutes the activation region of the proenzyme, thrombin, kallikreinor FXIIa are, in accordance with their substrate specificities, examplesof suitable physiological activators of the proenzyme.

Some of the properties of the novel protease which have been described,namely the fact that it can be isolated from plasma or from prothrombincomplex (PPSB) concentrates which are derived from plasma, theinhibition of its amidolytic activity by aprotinin and the describedmigration behavior in SDS-PAGE, both in the reduced and in thenon-reduced states, are reminiscent of a protease which was isolated byHunfeld et al. (Ann. Hematol. 1997; 74; A87, 113; Ann. Hematol. 1998;76; A101, P294 and Etscheid et al. Ann. Hematol. 1999, 78: A42) from aPPSB concentrate which was not defined in any more detail. In that case,the preparation was essentially achieved using an aprotinin matrix. As aresult of the amidolytic cleavage of certain peptide substrates, theactivity was described as being a thrombin-like activity. Hunfeld et al.did not find any influence on global clotting parameters such asprothrombin time, Quick or platelet aggregation.

The N-terminal sequencing of the protease described by Hunfeld et al.shows concordances with a protein whose cDNA was described by Choi-Miuraet al. (J. Biochem. 119: 1157-1165 (1996)). In its primary structure,the corresponding protein exhibits homology with an enzyme termedhepatocyte growth factor activating enzyme (HGFA).

When two bands which were isolated from SDS-PAGE under reducingconditions were subjected to N-terminal sequencing, the followingconcordances were established:

Molecular weight range of the band Amino acid sequence Author 10-35 kDaIYGGFKSTAGK (SEQ ID NO:1) present invention    30 kDa IYGGFKSTAG (SEQ IDNO:2) Hunfeld et al.    17 kDa IYGGFKSTAGKH (SEQ ID NO:3) Choi-Miura etal. 40-55 kDa LLESLDP (SEQ ID NO:4) present invention    50 kDa SLDP(SEQ ID NO:5) Hunfeld et al.    50 kDa SLLESLDPWTPD (SEQ ID NO:6)Choi-Miura et al.

Concordances are also found in other test results such as substratespecificity and the ability of the activity to be inhibited. Despitethis, it is still not possible at present to assume with confidence thatthese proteins are identical. At any rate, the previously investigated,abovementioned proteins have not been reported to possess the propertyof activating FVII or activating other factors (see below).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the level of FVII activity as the protease concentrationis increased while the incubation time remains constant.

FIG. 2 depicts the level of FVII concentration as the incubation time isincreased while the protease concentration remains constant.

FIG. 3 depicts the level of FVII activity as the concentration ofcalcium is increased.

On the basis of its described properties, the novel protease can be useddiagnostically and therapeutically.

1. Test Systems Using the Novel Protease

The novel protease can be used diagnostically in test reagents. Thus,the presence of factor VII can be determined qualitatively andquantitatively in a clotting test by adding the novel protease.

Conversely, the test system developed for measuring FVII activation canalso be used for detecting and quantifying the protease. For this, asolution containing the protease is mixed with an FVII-containingsolution and, after an appropriate incubation time, the resultingquantity of FVIIa is quantified. This can be carried out, for example,using the Staclot® FVIIa-rTF test (Stago/Boehringer Mannheim). When apreferred procedure is used, this test is not limited by the FVIIconcentration supplied. If the quantity of protease in the form of theproportion of total protein is known, which proportion can be determined

in a pure protease preparation, by means of the Kjeldahl method or bymeans of another protein assay with which the skilled person isfamiliar, or

using an antigen test for example based on specific antibodies and anappropriate immunochemical determination method such as ELISA, thespecific activity of the protease preparation can then be measured in acorresponding manner.

Surprisingly, a property has now been found in association withcharacterizing the protease further, which makes it possible to carryout an additional determination method. In association with incubationof the blood clotting factors VIII/VIIa and V/Va with said protease, andsubsequent quantification, it became clear that said clotting factorsare inactivated in a manner which is dependent on the proteaseconcentration and on the length of the incubation.

Another part of the subject matter of the invention is therefore a noveltest system for qualitatively and quantitatively detecting the proteasewhich activates blood clotting factor VII, in which system the proteasecan be determined by its action inactivating the blood clotting factorsVIII/VIIa or V/Va. This test system is based on a solution containingthe protease being incubated with factor VIII/VIIa or factor V/Va andthe remaining quantity of factor VIII/VIIa or the remaining quantity offactor V/Va being measured by means of a conventional activity test andthe amount of protease then being quantitatively determined from this bycomparison with a standard curve. In carrying out this test, theincubation of the protease activity is inhibited, after predeterminedperiods of time, by the limited addition of aprotinin, which has theadvantage that it has no effect, at these concentrations, on thesubsequent measurements of the test system. After that, the remainingactivities of the clotting factors are measured by means of a test whichis familiar to the skilled person. For this, a test system has, inparticular, proved its worth in which use is made of the so-calledCoamatic® factor VIII test (Chromogenix AB), which essentially containsfactors IXa and X, with the resulting amount of FXa being quantified, inthe presence of a thrombin inhibitor, by means of the conversion of achromogenic substrate (see last third of page 2). This amount is in turnproportional to the quantity of FVIII or FVIIIa. Determining theresidual FVIII activity then makes it possible to deduce theconcentration of protease which is present.

The degradation of the FVIII/FVIIIa or the FV/FVa due to the proteolyticeffect can be clearly demonstrated by SDS-PAGE Depending on the time forwhich the protease is incubated, for example, with an FVIII concentrate,bands which are typical for FVIII disappear while other, new bandsemerge or weak bands increase in intensity. Accordingly, the activity ofthe protease can also be correlated by quantifying the decreasing orincreasing bands and consequently measured quantitatively for exampleusing a protease standard. The changes in the band intensities on theSDS-PAGE electropherogram or following other electrophoretic methods canbe quantified, for example, using a scanner, with which a skilled personis familiar, and the appropriate program. In addition to this,anti-bodies against said clotting factors can be used for Westernblotting and employed for evaluation in the manner described. Antibodieswhich specifically detect the decreasing bands or, in particular, theemerging bands are particularly suitable. In this context, theseantibodies can also be used for establishing other immunochemical testssuch as an ELISA.

The proteolytic inactivation which has been described in the case ofFVIII/FVIIIa is also observed when the protease is incubated with factorV/Va, which exhibits a certain degree of structural homology with FVIII.The degradation can be monitored in suitable activity test systems andin SDS-PAGE/Western blotting.

Despite the inactivations of FV and FVIII, it was now found that addingthe protease to blood, to platelet-rich plasma or plasma shortened theclotting times, that is the procoagulatory effect predominated invarious so-called “global clotting tests”. These test systems areunderstood as being, for example, the non-activated partialthromboplastin time (NAPTT), the prothrombin time (PT) and therecalcification time. Since the shortening of these times, as measured,for example, in so-called coagulometers, by means of thromboelastographyor else in chromogenic tests, correlates with the concentration of aclotting-promoting substance, the concentration of the substance in asample can conversely be deduced using a calibration curve of theclotting time. The concentration of the “FVII activator” cancorrespondingly be determined using selected global clotting tests.

It was also surprising to find that the “FVII activator” is likewiseable to bring about effective activation of single chain urokinase(scuPA, single chain urokinase plasminogen activator) and single chaintPA (sctPA, single chain tissue plasminogen activator), that is can actas a plasminogen activator activator (PAA). The activity of theactivated PAs can be measured, for example, using chromogenicsubstrates. Accordingly, this property can therefore also be used fordetecting and quantifying the “FVII activator”. The activation of theplasminogen activators can also be determined in a coupled reaction inthe presence of plasminogen, either by the formation of plasmin itselfor by the dissolution of a fibrin clot which is brought about byplasmin.

In summary, therefore, it can be stated that the protease can be bothdetected and quantified by incubating it with a solution containingFVIII or FVIIIa and then determining the remaining quantity ofFVIII/VIIIa by means of a suitable activity test. In the same way, FV orFVa can be incubated with the protease and the remaining quantity ofFV/FVa can subsequently be quantified. The unknown proteaseconcentration can be determined quantitatively by comparison with astandard curve of increasing quantities of protease which is included inthe test. Various global clotting tests are likewise suitable for thequantification, with the protease concentration being read off acalibration curve on the basis of the shortening of the clotting time.The PAA activity of the protease can also be used for determinationpurposes.

Another feature of these tests is that the FV and FVIII inactivation andthe PAA activity are displayed particularly well in the presence ofadequately high concentrations of calcium, preferably >0.001 mM,particularly preferably >0.005 mM, e.g. in the form of CaCl₂. Incontrast to the direct chromogenic assay, in which, as has beendescribed above, both heparin and heparin-like substances and alsocalcium increase the protease activity, the inactivation of FV/FVIII isnot promoted, or only promoted insignificantly, by heparin. By contrast,the PAA activity is stimulated in the presence of both agents, that isby calcium and/or heparin or heparin-like substances.

The protease-mediated reactions can be very efficiently diminished orprevented by incubating the protease with inhibitors, particularlyantithrombin III in the presence of heparin or heparin-like substances(preferably in the presence of heparin), C1-esterase inhibitor,alpha2-antiplasmin, inter-alpha-trypsin inhibitor or known synthetic,low molecular weight protease inhibitors such as Guanidinocaproicacid-para-ethoxy-carbonylphenylester wich is available under thetrademark FOY®. These substances can therefore be used for stopping thereaction, in order, for example, to define incubation times precisely orto increase the specificity of the test still further. Decreasing thefree calcium ions in the mixture with a chelating agent, for example,can also be used for this purpose.

2. Stabilized Preparations of Factor V and Factor VIII

The further task now ensued, from the above-described observationsconcerning the proteolytic actions of the novel protease on clottingfactors V and VIII, of inhibiting the protease or reducing its activityin order to avoid losses of yield and the formation of what mightpossibly be interfering protein fragments. This is all the more relevantsince FV and FVIII are usually prepared from cryoprecipitates which havebeen obtained from plasma and in the presence of calcium ions becausethe latter are required for maintaining protein conformations.

Another part of the subject matter of the invention is therefore astabilized preparation of FV or FVIII which is free of the factor V orfactor VIII fragments formed due to proteolytic degradation as a resultof the fact that the protease activating the blood clotting factor VIIis inhibited. Since more detailed investigations have shown thatinactivation of factor V and factor VIII by said protease takes placeparticularly efficiently in the presence of calcium ion concentrationsgreater than 0.5 mM, the factor V or VIII preparation can be effectivelystabilized if, for the inhibition of the protease activating the bloodclotting factor VII, the concentrations of calcium ions in the factor Vor in the factor VIII preparation are adjusted to less than 1.0 mM,preferably to less than 0.5 mM. While the factor V- and factorVIII-inactivating properties of the protease are markedly reduced atthese concentrations, the quantity of calcium ions is still sufficientfor stabilizing the conformations of the FV and FVIII molecules. Theabovementioned quantities of calcium ions should not be exceeded, notmerely in the end product but also in the cryoprecipitate itself and inthe following purification steps.

In accordance with the above-described affinity of the protease or theproenzyme for heparin and heparin-like substances, the protease/theproenzyme can be removed from the FVIII- or FV-containing solution byincubating with immobilized heparin or other suitable immune- oraffinity-adsorbents. Polyclonal or monoclonal antibodies, respectiveantibody-fragments that are useful in preparing the immune adsorbentsare readily available by techniques known in the art in using all orpart of the protease or proenzyme as antigen.

However, natural or synthetic protease inhibitors can also be employed,where appropriate in addition to decreasing the quantity of calciumions, for preventing the proteolytic degradation of the FV or the FVIII.Proteins such as aprotinin, alpha2-antiplasmin, C1-esterase inhibitor orinter-trypsin inhibitor may be employed as inhibitors. Low molecularweight substances which are known to the skilled person as syntheticserine protease inhibitors can also be used in this context. Inhibitors,such as antithrombin III, whose inhibitory potential is increased byheparin or heparinoids can likewise be added. Thus, it has been found,surprisingly, that while heparin on its own is able to increase theamidolytic activity of the protease towards small chromogenicsubstances, it does not support inactivation of FV/FVIII.

3. Pharmaceuticals which Comprise the Novel Protease

The novel protease and/or its proenzyme can also be usedtherapeutically.

They can be employed as a blood coagulating agent, either on their ownor together with substances which increase the activity of the protease,such as heparin, or heparin-related substances, such as heparan sulfate,and/or calcium ions, with it being possible additionally to add factorVII as well, in its inactive form, to this agent. The use of such anagent, in which its FVIII-bypassing activity (FEIBA) is exploited, forexample, can be indicated when intolerances exist toward FVIII and/orFIX and/or FXI and/or the contact phase proteins, such as FXII, forexample on account of the presence of antibodies, or when other types ofdeficiency situation exist. In this connection, the FVII can beactivated either in vitro, in the plasma, in enriched fractions or byacting on purified FVII. It is also possible to use the novel bloodcoagulating agent ex vivo for general hemorrhage prophylaxis or forstaunching hemorrhages.

On the other hand, the observed inhibition of the novel protease byaprotinin or the abovementioned inhibitors can be used for developing anagent which comprises a protease inhibitor and which diminishes theability of the blood to coagulate. In addition to this, the novelprotease can also be used to identify physiological or non-physiologicalfactors, such as synthetic peptides, which impair blood clotting becauseof their protease-inhibiting effect. The peptide sequences of thechromogenic substrates which are transformed particularly efficiently,such as those of the S 2288 (see above for details), can be used as astructural basis for this. The addition of suitable inhibitors toclotting preparations, or during their preparation, can be necessary ifthese preparations are to be free of proteolytic activities.

Surprisingly, a property has now been found, in association withcharacterizing the protease further, which opens up the possibility ofan additional use for the so-called “factor VII activator” protease.When single chain plasminogen activators such as prourokinase (singlechain urokinase, scuPA, single chain urokinase plasminogen activator) orsctPA (single chain tissue plasminogen activator) are incubated, the“factor VII activator” brings about activation of these plasminogenactivators (PA). In this connection, there is a limited proteolysis ofthe single chain PAs, resulting in the formation of double chainproteases, which are particularly suitable for activating plasminogen.The resulting plasmin is the effector of fibrinolysis, that is thephysiological system which is responsible for dissolving thrombi. PAs,such as prourokinase or tPA, are endogenous proteins which are releasedwhen needed and which, as is known, are activated by plasmin or bykallikrein (scuPA). The mechanism by which scuPA is activated in thehealthy state has not yet been fully clarified.

The plasminogen activators are employed therapeutically, as isolated orrecombinantly prepared proteins, in pharmaceutical preparations inassociation with thromboembolic diseases or complications, such as inleg vein thrombosis, cardiac infarction or strokes.

In accordance with the properties of the “factor VII activator” whichhave now been found, the latter can be used for in vivo or ex vivoactivation of plasminogen activators such as prourokinase or sctPA. Thisactivity can also be applied by using said protease for the prophylaxisor therapy of thromboembolic diseases, specifically in combination withsingle chain or double chain plasminogen activators or anticoagulants aswell. This possible use is not contradictory to the fact that theprotease is also able to act in a procoagulatory manner. The question ofwhich of the two reactions predominates is probably resolved by theavailability of the physiological substrates. According to the currentstate of knowledge, factor VII is activated moderately in plasma andcontinuously maintains a certain concentration of FVIIa in order to beable to counteract immediately any sudden vascular damage. On the otherhand, only nanogram quantities of tissue plasminogen activator andurokinase plasminogen activator are present in a milliliter of bloodplasma. It is only when fibrin deposition or thrombi occur that there isan increase in the concentration, by secretion or synthesis, ofplasminogen activators, which then display their thrombolytic activityby activating plasminogen after they have been activated locally, inparticular when bound to the thrombus. When single-chain PAs arepresent, particularly in a locally restricted manner, their activationmight outweigh FVII activation, thereby making it possible to adjust tothe physiological situation. Accordingly, this protease might alsoregulate hemostasis, thereby indicating a replacement with the proteaseand/or the proenzyme in the case of inborn and acquired deficiencystates.

Another part of the subject matter of the invention is therefore apharmaceutical preparation which comprises a quantity of the bloodclotting factor VII-activating protease and/or its proenzyme form whichis sufficient for dissolving fibrin-containing thrombi. This preparationmay additionally comprise single chain plasminogen activators (PA)and/or anticoagulants. When the proenzyme is present it is advantageousto comprise a suitable activating agent within or together with thepharmaceutical preparation above.

Since it has been found that the plasminogen activator-reinforcingeffect of the “FVII activator” is particularly promoted by calciumand/or heparin and heparin-like substances such as dextran sulfate,pharmaceutical preparations which additionally comprise soluble calciumsalts and/or heparin or heparin-like substances may particularlyadvantageously be employed for dissolving, in accordance with theinvention, fibrin-containing thrombi. In this context, theprotease/proenzyme can be employed on its own or in combination withsingle chain or double chain plasminogen activators with or withoutsubstances which exhibit particular affinities for the protease andthereby increase its activity as carrier substances for prolongingplasma half life or as mediators to surfaces.

Pharmaceutical preparations which comprise the blood clotting factorVII-activating protease can, because of its special fibrinolytic effect,be employed for treating diseases which are caused by fibrin-containingthrombi. Fibrinolytic processes are also involved in wound healingprocesses. In this connection, said protease and/or proenzyme can beadministered intravenously or locally, subcutaneously, intradermally orintramuscularly, or else topically in the case of injuries and wounds,or bound to a suitable carrier matrix. Both protease/proenzyme which hasbeen isolated from body fluids such as blood or plasma andprotease/proenzyme which has been prepared recombinantly ortransgenically can be employed in this context. The protease/proenzymeis also suitable for use as a component of a so-called fibrin adhesive,which should not then contain any substance, such as aprotinin, whichinhibits the protease/proenzyme. In this case, use can be made of theclotting-shortening properties of the protease.

The protease/proenzyme above may be used for inherited or acquiredhemostasis deficiencies, in (diffuse) bleeding occurencies respectivethrombosis associated complications. If used to treat bleeding thecombination of protease/proenzyme together with F VIII optionally underaddition of further clotting factors is advantageous.

4. Process for Pasteurizing the FVII-activating Protease

As a protein which has been isolated from human plasma, the novelprotease and/or its proenzyme can only be employed as a pharmaceuticalpreparation if it has previously been subjected to a process forinactivating viruses. The pasteurization process is in particularrecognized as being the most important process for inactivating viruses.However, heating at about 60° C. for up to 10 hours requires the proteinwhich is to be treated to be of adequate stability. The optimalstabilizers have to be determined separately for each protein and theirconcentrations have to be optimized.

In the case of the novel protease and/or its proenzyme, conditions whichstabilize the protein in solution, without any pasteurization beingperformed, have already been mentioned above. In this regard, a slightlyacidic pH range has in particular proved to be advantageous. However,when a pasteurization is carried out under these conditions, the novelprotease and/or its proenzyme as a rule loses more than 50% of itsoriginal activity.

It has now been found that a pasteurization of a pharmaceuticalpreparation comprising the novel protease and/or its proenzyme ensuresoptimal stabilization results if the preparation is prepared

a) in a pH range of from 3.5 to 8.0, preferably in a pH range of from4.0 to 6.8;

b) in the added presence of one or more amino acids in a quantity ofmore than 0.01 mol/l, preferably more than 0.05 mol/l; and/or

c) in the added presence of a sugar or of a combination of differentsugars having a total concentration of more than 0.05 g/ml, preferablymore than 0.2 g/ml; and/or

d) in the added presence of one or more substances which are able tocomplex calcium ions, such as citrate, oxalate, ethylenediaminetetraacetic acid, etc.

Additives such as albumin, Haemaccel®, heparin and heparinoids,glycerol, glycol and polyethylene glycol, may also be used separately ormixed together. After the pasteurization has been completed, the sugars,amino acids and other additives which have been added as stabilizers canbe decreased, or removed completely from the preparation, using methodswith which the skilled person is familiar. The results of thepasteurization processes are given in Examples 12 and 13.

EXAMPLE 1

The Staclot® FVIIa-rTF test system (Stago/Boehringer Mannheim) was usedfor demonstrating activation of FVII by the prepared protease. Thisdetection system is based on the particular property of (recombinant)soluble tissue factor (rTF) which is only able to use the preformedactivated FVII (FVIIa) for initiating the extrinsic clotting pathway. Incontrast to the situation when complete tissue factor is used, thismakes it possible to determine the real content of FVIIa precisely.

Isolated FVII (Enzyme Research Labs) was used for the activationexperiments. This FVII itself contains traces of FVIIa since it isisolated from human plasma. The concentration was adjusted to 0.05 IU ofFVII/ml by diluting with buffer. The FVII was incubated at roomtemperature for 10 min with the test substances and then tested for thetrue FVIIa content. The FVIIa contents were quantified using a referencecurve which was constructed in parallel.

It was ascertained in preliminary experiments, which are not describedhere, that while, in the concentration employed, aprotinin completelyinhibited the activity of the prepared protease, it had no direct effecton the FVIIa nor any significant effect on the FVIIa-rTF test system.

The results given below relate in each case to triplicatedeterminations.

The following experimental assays were accordingly set up:

1. FVII:

Result: 10 mlU of FVIIa/ml

Non-activated FVII was used as the control assay. This already containstraces of FVIIa (see above) in the order of magnitude of 10 mlU ofFVIIa/ml.

2. FVII+aprotinin:

In this assay, FVII was incubated in the presence of aprotinin and usedin the FVIIa-rTF assay in order to demonstrate that FVIIa itself was notinhibited, and nor was the test affected by the aprotinin employed. Thiswas confirmed (in comparison with assay 1).

3. Protease+FVII (incubation), followed by the addition of aprotinin:

Result: 18 mlU of FVIIa/ml

In this case, the protease was given time to activate FVIIa. Aprotininwas only added, in order to inhibit the protease, after the 10-minuteincubation had taken place. The resulting FVIIa was quantified in theFVIIa-rTF assay. Subtracting the FVIIa base value (assay 1), 8 mlU ofFVIIa/ml have therefore been formed by the action of the protease underthe chosen conditions.

4. Protease+aprotinin, followed by the addition of FVII

Result: 11 mlU of FVIIa/ml

In this assay, the protease was inhibited with aprotinin before contactwith FVII. Neither the subsequent incubation with FVII, nor thefollowing FVIIa quantification led to any significant increase in theFVIIa content (because of the range of variation in the assay, 11 versus10 mlU/ml in assay 1 is not to be regarded as being significant).

5. Protease

Result: 0 mlU of FVIIa/ml

This assay demonstrated that, at the concentration selected, theprotease did not itself have any effect on the FVIIa-rTF test system.

In summary, it follows from the above that

the described protease activates FVII;

the activation of FVII by the protease takes place “directly”, that isindependently of the presence of rTF;

the activation of FVII can be inhibited by aprotinin; at theconcentration selected, aprotinin itself does not have any significantinfluence on the test system.

EXAMPLE 2

This example describes how FVII is activated in a reaction which isdependent on the concentration of the protease and on the time overwhich the protease is incubated with FVII.

Test systems and reagents were selected to correspond with theconditions described in Example 1. In a first series of experiments, theinitally introduced FVII was preincubated with different dilutions (1:5,1:10 and 1:20) of the protease-containing solutions (5 min at RT), thentreated with aprotinin (to inhibit the protease) and subsequently testedfor its content of FVIIa in the FVIIa-rTF assay.

Once again, the parallel assays, in which the protease had beeninhibited by aprotinin before contact with FVII, served as controlassays.

The results are given as activation factors, i.e. correspond to x timesthe value which was measured in the abovementioned control assay:

Assay Control Protease + FVII Protease + aprotinin Incubation +Incubation + Aprotinin FVII Dilution of the protease solution Activationfactor 1:5 2.6 1.0 1:10 2.0 1.0 1:20 1.6 1.0

The activation factor 1:0 of the control assays corresponds to thecontrol, which was additionally included and in which only the testbuffer, containing the FVII employed, was treated under identicalincubation conditions and tested. That is, no significant activationtook place in the control assays.

It follows from this that FVII is activated by the protease in a mannerwhich is dependent on the concentration of the protease.

It was similarly demonstrated that, when the concentrations of thecoreactants are kept constant, the FVII is activated by the protease ina manner which is dependent on the length of the incubation.

When equal volumes of a solution containing 0.2 lU of FVII/ml and a1:10-diluted protease solution were incubated together, the followingcontents of FVIIa were obtained after incubating for the relevant timesand subsequently adding aprotinin (in order to stop the activation):

Length of incubation Activation factor 0 min 1.0 2.5 min 1.3 5.0 min 2.010.0 min 2.8 40.0 min >3.8

It follows from this that FVII is activated by the protease in atime-dependent manner.

EXAMPLE 3

Using this example, it will be demonstrated that activation of FVII bythe protease is increased in the presence of calcium ions and heparin.

25 μl of the protease-containing solution were mixed with 50 μl of

buffer (control)

15 mM CaCl₂

50 USP units of heparin/ml

Pathromtin (lipid mixture, aliquot dissolved in accordance with themanufacturer's instructions) at room temperature for 5 min, and thentreated with 150 μl of a tris/NaCl buffer solution (pH 8.2) and 25 μl ofthe chromogenic substrate S2288 (3 mM); the time-dependent change in theextinction at 405 nm was then measured (at 37° C.). The activationfactors, related to the buffer control (x times), are given in thefollowing table.

Activation factor Assays (x times buffer control) Buffer control 1.0+CaCl₂ 3.6 +Heparin 2.6 +Lipid 0.9 +CaCl₂ + heparin 4.3 +CaCl₂ + lipid3.3 +Heparin + lipid 2.7 +CaCl₂ + heparin + lipid 3.7

Under the conditions used in this example, marked increases in theactivity of the protease can be observed in the presence of calcium ionsand/or heparin.

EXAMPLE 4

In each case, 25 μl of a solution, containing 10, 1 or 0.1 μg ofprotease/ml, were mixed with 25 μl of FVIII (2 lU/ml), after which 25 μlof CaCl₂ (25 mM) and 25 μl of Pathromtin® (Dade Behring GmbH) wereadded. After incubating at 37° C. for 0, 3, 10 and 20 min, the reactionwas stopped by adding 400 μl of aprotinin (500 KlU/mg). A sample inwhich aprotinin was introduced initially served as a control.

Each sample was diluted in tris-buffer/BSA. In each case, 50 μl of thissolution were mixed with 50 μl of the factor reagent (essentiallycomposed of FIXa, FX and a thrombin inhibitor, appropriately modified inaccordance with the Coamatic® FVIII test, Chromogenix AB) and incubatedat 37° C. for 10 min. After 50 μl of substrate (e.g. S 2765,N-a-Cbo-D-Arg-Gly-Arg-pNA) had been added, the reaction was stoppedafter a predetermined period of incubation by adding 50 μl of aceticacid (50%), and the OD405 nm was then measured. A standard curve forFVIII was used for determining the concentration in the sample.

Results

In a first assay, the time for which the protease was incubated withFVIII (2 lU/ml) was kept constant (10 min) but the concentration of theprotease was varied (0.1, 1 and 10 μg/ml). The reaction was stopped andthe residual concentration of active FVIII was determined. As theprotease concentration increased, correspondingly more FVIII wasinactivated (FIG. 1).

The protease content of a sample can be quantified using an appropriatestandard curve.

In a second assay, the concentration of the protease was kept constant(10 μg/ml) but the time of incubation with FVIII (2 lU/ml) was varied. Amarked reduction in the residual concentration of active FVIII was seenas the length of incubation increased (FIG. 2).

EXAMPLE 5

The influence of the “FVII activator” on the activity of factor V wasinvestigated:

25 μl protease-containing solution (0-100 μg/ml) were incubated with 50μl of FV (5 lU/ml) and 25 μl of 25 mM CaCl₂ (0-20 min) and, after that,400 μl of buffer containing 100 KlU of aprotinin/ml were added.

In each case, 100 μl of each incubation assay were then incubated with100 μl of V-deficient plasma at 37° C. for 1 min, after which 200 μl ofThromborel S® were mixed in and the clotting times were determined in aSchnitger and Gross coagulometer. The residual activities of FV weredetermined.

Results

Residual FV activity Time for which protease Protease concentrationincubated with FV (min) (μg/ml) 0 10 20 10 93 91 100 30 100 93 28 100100 29 13

This example demonstrates that FV was inactivated by the protease overtime.

EXAMPLE 6

The influence of the “FVII activator” on clotting times in so-calledglobal tests was investigated using Schnitger and Gross coagulometers.All the difference values listed correspond to the clotting times whichwere shortened by this amount.

NAPTT (Non-activated Partial Thromboplastin Time)

The protease-containing solution was diluted with buffer down to 100,30, 10 and 3 μg/ml. 100 μl of each of these solutions were incubated, at37° C. for 2 min, with 100 μl of citrate plasma (standard human plasmapool or individual donors) and 100 μl of Pathromtin®, after which 100 μlof 25 mM CaCl₂ were added; the clotting times were then determined. Thedifferences between these measured values and the corresponding clottingtimes obtained with buffer solution instead of the protease weredetermined.

Clotting time differences (buffer minus sample) (sec) Proteaseconcentration (μg/ml) Sample No. 0 3 10 30 100 Standard human 0 13 20 4243 plasma (213 sec) 1 0 20 33 42 41 2 0 27 31 45 47 3 0 13 14 23 29 4 018 37 51 50 5 0 25 49 54 46

The addition of FVII-acitvator resulted in a concentration dependentshortening of NAPTT.

Plasma Recalcification Time

The protease-containing solution was diluted with buffer down to 100,30, 10 and 3 μg/ml. 100 μl of each of these solutions were incubatedwith 100 μl of citrate plasma (standard human plasma pool or individualdonors) at 37° C. for 1 min, after which 100 μl of 25 mM CaCl₂ wereadded; the clotting times were then determined. The differences betweenthese measured values and the corresponding clotting times obtained withbuffer solution instead of protease were determined.

Clotting time differences (buffer minus sample) (sec) Proteaseconcentration (μg/ml) Sample No. 0 3 10 30 100 Standard human 0 17.215.1 30.5 50.4 plasma (283 sec) 1 0 29.8 51.7 60.3 90.1 2 0 25.2 51.769.5 101.3 3 0 28.0 — 39.0 74.6 4 0 27.3 42.7 55.6 91.8 5 0 44.3 69.1101.2 134.2

PT (Prothombin Time)

The protease-containing solution was diluted with buffer down to 100,30, 10 and 3 μg/ml. 100 μl of each of these solutions were incubatedwith 100 μl of citrate plasma (standard human plasma pool or individualdonors) 37° C. for 1 min, after which 200 μl of Thromborel S® (DadeBehring GmbH) were added; the clotting times were then determined.

The differences between these measured values and the correspondingclotting times obtained with buffer solution instead of protease weredetermined.

Clotting time differences (buffer minus sample) (sec) Proteaseconcentration (μg/ml) Sample No. 0 3 10 30 100 Standard human 0 1.0 1.71.5 2.4 plasma (13.6 sec) 1 0 0.7 1.3 2.4 2.7 2 0 0.3 0.4 1.7 3.1 3 00.4 0.7 1.5 1.8 4 0 0.1 0.7 1.8 3.1 5 0 0.3 0.5 1.2 2.8

The clotting times in the above global tests were shortened in a mannerwhich was dependent on the concentration of the protease. In acorresponding manner, it was possible, after “calibrating” a plasmawhich was used with a known quantity of the “FVII activator”, todetermine the protease concentration in a sample by reading off from astandard curve.

EXAMPLE 7

The plasminogen activator-activating properties of the “FVII activator”were investigated using single chain urokinase (scuPA) and single chaintPA (sctPA).

Assay

0.1 ml of PA solution (20 μg of scuPA/ml or 100 μg of sctPA/ml)

+0.1 ml of test buffer or 100 U of heparin/ml in test buffer or 20 mMCaCl₂ in the test buffer

+0.5 ml of test buffer

+0.1 ml of protease/sample (increasing concentrations: 2-10 μg ofscuPA/ml or 50-200 μg of sctPA/ml)

Incubation at 37° C.

+0.1 ml of 100 KlU of aprotinin/ml in test buffer

Incubation at 37° C. for 2 min

+0.1 ml of substrate S-2444 (3 mM)

As a control, aprotinin was introduced initially, instead of theplasminogen activator (PA), prior to the first incubation, and carriedthrough in each case. In return, PA was not added until later, in placeof the aprotinin.

The Difference of the measurements (Δ) ΔOD_(405 nm) was determinedphotometrically. The control values which were obtained were subtractedfrom the sample/protease values and in this way the PA activity whichwas caused by the PAA activity was determined (in mlU/min).

Results

scuPA Activation (20 μg of scuPA/ml, 2-10 μg of “FVII Activator”/ml)

A. Stimulant: none

Resulting PA activity (Δ mlU/min) Incubation time “FVII activator”(μg/ml) (min) 2 5 10 2 25 60 117 5 79 179 165 10 186 449 517 B.Stimulant: heparin 2 190 332 425 5 330 455 458 10 417 462 460 C.Stimulant: CaCl₂ 2 255 370 401 5 338 424 438 10 416 445 448

The tables illustrate the fact that scuPA was activated in a mannerwhich was dependent on the concentration of the “FVII activator” and onthe length of the incubation. At the same time, both heparin and calciumhad a stimulatory effect on the activation of the PA which was broughtabout by the protease.

sctPA Activation(100 μg of sctPA/ml, 50-200 μg of “FVII Activator”/ml)

Since the turnover rate of the activated tPA only increases by a factorof 3-4 compared with the tPA proenzyme (while that of uPA increases by afactor of 1000-1500), higher concentrations of the two coreactants (seeabove) had to be selected in order to obtain an analyzable measurementsignal.

Incubation time Resulting PA activity (Δ mlU/min) (min) “FVII activator”(200 μg/ml) 1 10.2 2 16.8 5 38.8 10 60.2 20 73.3

B. Dependence on the concentration of the “FVII activator” (incubationtime: 20 min, at 37° C.), stimulant: heparin (100 lU/ml)

“FVII activator” (μg/ml) PA activity (Δ mlU/min) 50 33.6 100 51.0 20071.9

C. Stimulants (period of incubation: 20 min, at 37° C.)

Stimulant PA activity (Δ mlU/min) None 5.9 CaCl₂ 25.3 Heparin 63.8

The tables demonstrate that sctPA was also activated in a manner whichwas dependent on the concentration of the protease and on the incubationtime. Both heparin and calcium ions had a stimulatory effect on thePA-activating ability of the “FVII activator”.

EXAMPLE 8

Two FVIII-containing solutions, one of which was essentially free of vonWillebrand Factor while the other contained vWF, were incubated with theabovementioned protease in the presence of calcium. After predeterminedtimes, the residual FVIII activities were determined by means of achromogenic test and related to the control assays without protease.

For this, 25 μl of a solution containing 0.1 lU of FVIII/ml were treatedwith the same volume of the protease solution (10 μg/ml) and the wholewas mixed with 25 μl of CaCl₂ (25 mM). After incubation periods of 0, 5,10 and 20 min at 37° C., the assays were in each case treated with 400μl of a solution containing 200 KlU of aprotinin/ml in order to stop theproteolytic activity of the protease. Preliminary experiments had shownthat this concentration of aprotinin had no significant interferingeffect on the FVIII activity test described below (assays 1+3). In assay2, the protease was incubated with aprotinin prior to contact withFVIII, after which the procedure was as described above.

In each case, 50 μl of the stopped sample (or after further dilution)were then treated with the so-called factor reagent, essentiallycomposed of FIXa, FX and a thrombin inhibitor, and incubated at 37° C.for 10 min. Following the addition of 50 μl of a chromogenic substratewhich is cleaved by activated FX, the reaction was stopped after 5minutes of incubation by adding 50 μl of acetic acid (50%); theΔOD_(405 nm) was then measured. The FVIII activity (mlU) was ascertainedwith the aid of a standard curve which was constructed using a dilutionseries which was prepared from the FVIII concentrate and which wasincluded in the test.

The FVIII activities are given in percentages of the controls to whichprotease was not added.

Results: FVIII activity (%) Incubation period (min) Assay 0 5 10 20 1.FVIII 97 27 11 <1 2. FVIII/aprotinin 98 97 97 96 3. FVIII/vWF 98 16 14 1

In the presence of CaCl₂ (in this case 6.25 mM), FVIII was inactivatedby the protease in a manner which was dependent on the length of theincubation. The vWF did not protect the FVIII from inactivation by theprotease. Inhibition of the protease with aprotinin prior to contactwith FVIII prevented the latter from being inactivated.

EXAMPLE 9

This experimental series was carried out as described in example 1/assay1, but in this case the concentrations of calcium in the mixtures ofprotease and FVIII were varied. For this, CaCl₂ was added, from thestock solution of calcium, up to the final concentrations shown in FIG.3.

Results

If the concentration of calcium in the assay is decreased below 1 mM,approx. 50% of the FVIII is then spared under these conditions. Below0.5 mM calcium, the percentage spared is more than 60% (FIG. 3).

EXAMPLE 10

The influence of the “FVII activator” on the clotting times in so-calledglobal tests was investigated by means of thromboelastography.

The change in the shear elasticity or the strength of the relevant bloodclot was recorded continuously using a Hilgard TEG meter (from Hellige).The so-called r and k values are, respectively, the times from thebeginning of blood withdrawal and from the start of the clottingreaction, and, in the case of citrate blood plasma, the time ofrecalcification until the TEG curve has been broadened by 1 mm and thetime from the endpoint of the r value until the curve has been broadenedto 20 mm (clot formation time).

For this, aliquots of 150 μl of blood or plasma from 5 donors were ineach case incubated in the measuring cuvettes at 37° C. for 2 min, afterwhich 50 μl of sample (protease) were mixed in. The reaction was startedby adding 100 μl of 25 mM CaCl₂. The final concentration of the “FVIIactivator” in the assay was 15 μg/ml. The shortening of the r time wasmeasured in relation to the assay which contained buffer instead of thesample.

Results: r time k time r + k time Blood No. Sample (min) (min) (min) 1Protease 5.2 3.4 8.6 1 Buffer 7.8 5.6 13.4 2 Protease 5.2 5.1 10.3 2Buffer 6.8 7.1 13.9 3 Protease 4.0 5.2 9.2 3 Buffer 6.5 6.3 12.8 4Protease 4.5 4.8 9.3 4 Buffer 4.8 6.0 10.8 5 Protease 4.2 3.8 8.0 5Buffer 7.0 5.8 12.8 Plasma No. Sample r time (min) 1 Protease 9.0 1Buffer 11.3 2 Protease 9.2 2 Buffer 12.5 3 Protease 9.5 3 Buffer 9.6 4Protease 8.2 4 Buffer 12.1 5 Protease 9.7 5 Buffer 14.1

This example makes clear that, in almost all cases, addition of theprotease resulted in a marked shortening of the clotting time. In thispresent instance, the-fibrinolytic properties of the “FVII activator”receded into the background. A reason for this is that in “normalsubjects”, the concentrations of plasminogen activator in the plasma liein the nanogram region and do not have any effect in the in-vitroclotting test.

EXAMPLE 11

The FVIII-bypassing activity of the protease was demonstrated by thefollowing experimental assay: thromboelastography was used as themeasuring technique. The r time was evaluated (see Example 10). A sampleof whole blood was incubated with a monoclonal antibody, whoseFVIII-activity-inhibiting properties were known, in order to simulatethe presence of a naturally occurring FVIII inhibitor (antibody againstFVIII). This sample was compared with the whole blood sample control(buffer instead of Mab). The FEIB activity of the protease was tested byadding the protease (final concentration 17 μg/ml) to the whole bloodsample which had been inhibited by the Mab. Protease was added to afurther sample, and the effect of the protease, on its own, on the rtime was determined.

Results: r time Whole blood control 8.0 Whole blood + mAb 11.0 Wholeblood + mAb + protease 8.0 Whole blood + protease 3.5

The lengthening of the r time, caused by the anti-FVIII mAb, wasnormalized once again by the presence of the protease, therebyillustrating the FEIB activity of the protease. On its own, the proteaseshortened the clotting time, as already demonstrated above.

EXAMPLE 12

The following substances were added to a solution, which contained 50 μgof the FVII-activating protease/ml, to give the corresponding finalconcentrations:

25 mM Na citrate

25 mM HEPES

100 mM arginine

0.75 g of sucrose/ml

The solution was divided into portions and the aliquots were in eachcase adjusted to different pH values of from 5.0 to 8.6 and then heatedat 60° C. for 10 hours.

The activities of the heated protease solutions were determined in achromogenic test, with the time-dependent amidolysis of the chromogenicsubstrate S2288 (H-D-lle-Pro-Arg-pHA×2 HCl, Chromogenix AB, Sweden)being recorded. This activity was expressed as a percentage of thealiquots which were unheated and were measured in parallel:

Results: Assay Activity (%) Starting material 100 pH 5.0 76 pH 5.5 65 pH6.1 81 pH 6.5 50 pH 7.1 43 pH 7.5 46 pH 8.1 46 pH 8.6 32

This series of experiments makes clear that the stabilization,particularly in the acid pH range, has markedly reduced the inactivationof the protease. The slight “breakthrough” at pH 5.5 can be explained bythe fact that the isoelectric point of the protease is in this range. Nacitrate prevents a loss of activity of >50% occurring in the preferredpH range.

Example 13

The assay at pH 6.1 (Example 1) showed the best stabilization of theprotease. Accordingly, different additives were tested at pH 6.0 andevaluated as described in Example 1:

The following final concentrations were set, with the concentration ofthe protease being 50 μg/ml:

50 mM Na citrate/50 mM NaCl, pH 6.0

0.75 g of sucrose/ml

100 mM glycine

100 mM arginine

Results: Assay Activity (%) Starting material 100 Na citrate/NaCl 54 Nacitrate/NaCl/sucrose 85 Na citrate/NaCl/sucrose/glycine 92 Nacitrate/NaCl/sucrose/arginine 97

Marked stabilization of the protease was demonstrated by adding sucroseand in each case one amino acid.

6 1 11 PRT Unknown Description of Unknown Organism Illustrative peptide1 Ile Tyr Gly Gly Phe Lys Ser Thr Ala Gly Lys 1 5 10 2 10 PRT UnknownDescription of Unknown Organism Illustrative peptide 2 Ile Tyr Gly GlyPhe Lys Ser Thr Ala Gly 1 5 10 3 12 PRT Unknown Description of UnknownOrganism Illustrative peptide 3 Ile Tyr Gly Gly Phe Lys Ser Thr Ala GlyLys His 1 5 10 4 7 PRT Unknown Description of Unknown OrganismIllustrative peptide 4 Leu Leu Glu Ser Leu Asp Pro 1 5 5 4 PRT UnknownDescription of Unknown Organism Illustrative peptide 5 Ser Leu Asp Pro 16 12 PRT Unknown Description of Unknown Organism Illustrative peptide 6Ser Leu Leu Glu Ser Leu Asp Pro Trp Thr Pro Asp 1 5 10

What is claimed is:
 1. A protease which a) activates blood clotting factor VII, b) is inhibited by the presence of aprotinin, c) is increased in its activity by the presence of at least one of the following: calcium ions, heparin, or heparin related substances, and d) in SDS-PAGE, on subsequent staining in the non-reduced state, comprises one or more bands in the molecular weight range from 50 to 75 kDa; and in SDS-PAGE, on subsequent staining in the reduced state, comprises a band at 40 to 55 kDa, one or more bands in the molecular weight range from 10 to 35 kDa, and a band, which corresponds to a proenzyme, in the molecular weight range between 60 and 65 kDa.
 2. The protease as claimed in claim 1, wherein the band obtained in SDS-PAGE in the reduced state in the molecular weight range from 60 to 65 kDa and from 40 to 55 kDa has an amino acid sequence of Leu-Leu-Glu-Ser-Leu-Asp-Pro (SEQ ID NO: 4), and the band obtained in the molecular weight range from 10 to 35 kDa has an amino acid sequence of Ile-Tyr-Gly-Gly-Phe-Lys-Ser-Thr-Ala-Gly-Lys (SEQ ID NO: 1).
 3. The protease as claimed in claim 1, whicih is obtained by fractionation of blood plasma or of prothrombin complex (PPSB) concentrates.
 4. The proenzyme as claimed in claim 1 which in SDS-PAGE in the reduced state has a band in the molecular weight range between 60 and 65 kDa and contains the amino acid sequences Leu-Leu-Glu-Ser-Leu-Asp-Pro (SEQ ID NO: 4), an Ile-Tyr-Gly-Gly-Phe-Lys-Ser-Thr-Ala-Gly-Lys (SEQ ID NO: 1).
 5. A process for obtaining or removing a protease or proenzyme comprising: obtaining the protease or proenzyme from blood plasma or prothrombin (PPSB) concentrates after anion exchange chromatography by means of affinity chromatography using heparin or a substance related to heparin or dextran sulfate; or removing the protease or proenzyme from blood plasma or prothrombin (PPSB) concentrates after anion exchange chromatography by means of affinity chromatography using heparin or as substance related to heparin or dextran sulfate, wherein the protease is the protease as claimed in any one of claims 1 to 3 and the proenzyme in SDS-PAGE in the reduced state has a band in the molecular weight range between 60 and 65 kDa and contains the amino acid sequences Leu-Leu-Glu-Ser-Leu-Asp-Pro (SEQ ID NO: 4), and Ile-Tyr-Gly-Gly-Phe-Lys-Ser-Thr-Ala-Gly-Lys (SEQ ID NO: 1).
 6. A reagent for diagnostic purposes comprising at least one of the following: the protease as claimed in any one of claims 1 to 3 and a proenzyme in SDS-PAGE in the reduced state having a band in the molecular weight range between 60 and 65 kDa and containing the amino acid sequences Leu-Leu-Glu-Ser-Leu-Asp-Pro (SEQ ID NO: 4), and Ile-Tyr-Gly-Gly-Phe-Lys-Ser-Thr-Ala-Gly-Lys (SEQ ID NO: 1).
 7. A reagent for the detection of factor VII comprising at least one of the following: the protease as claimed in any one of claims 1 to 3 and a proenzyme in SDS-PAGE in the reduced state having a band in the molecular weight range between 60 and 65 kDa and containing the amino acid sequences Leu-Leu-Glu-Ser-Leu-Asp-Pro (SEQ ID NO: 4), and Ile-Tyr-Gly-Gly-Phe-Lys-Ser-Thr-Ala-Gly-Lys (SEQ ID NO: 1); optionally together with compounds which enhance the activity of the protease.
 8. A stabilized solution comprising at least one of the following: the protease as claimed in any one of claims 1 to 3 or a proenzyme in SDS-PAGE in the reduced state having a band in the molecular weight range between 60 and 65 kDa and containing the amino acid sequences Leu-Leu-Glu-Ser-Leu-Asp-Pro (SEQ ID NO: 4), and Ile-Tyr-Gly-Gly-Phe-Lys-Ser-Thr-Ala-Gly-Lys (SEQ ID NO: 1); wherein the stabilized solution is adjusted to a pH of 4.0 to 9.0 by addition of a buffer and optionally contains ethylene glycol or glycerol in an amount 5-80% by weight.
 9. A reagent for analytical purposes comprising at least one of the following: the protease as claimed in any one of claims 1 to 3 and a proenzyme in SDS-PAGE in the reduced state having a band in the molecular weight range between 60 and 65 kDa and containing the amino acid sequences Leu-Leu-Glu-Ser-Leu-Asp-Pro (SEQ ID NO: 4), and Ile-Tyr-Gly-Gly-Phe-Lys-Ser-Thr-Ala-Gly-Lys (SEQ ID NO: 1).
 10. A reagent for diagnostic and analytical purpose comprising at least one of the following: the protease as claimed in any one of claims 1 to 3 and a proenzyme in SDS-PAGE in the reduced state having a band in the molecular weight range between 60 and 65 kDa and containing the amino acid sequences Leu-Leu-Glu-Ser-Leu-Asp-Pro (SEQ. ID. NO. 4), and Ile-Tyr-Gly-Gly-Phe-Lys-Ser-Thr-Ala-Gly-Lys (SEQ ID NO: 1).
 11. The reagent as claimed in claim 7 wherein the compounds which enhance the activity of the protease are selected from calcium ions, heparin, heparin sulfate, and heparin-related substances. 